Substituted tetrahydro pteridines and method of preparing the same



United States Patent SUBSTITUTED TETRAHYDRO PTERIDINES AND METHOD OF PREPARING THEiSAME 1 16 Claims. erase -281.511.

This invention relates to new substituted tetrahfydro pteridines having biological activity and to'processes of preparing the same. Q

In 1948 Sauberlich and Baumann, Journal of Biological Chemistry, 176, page 165 (1948), recognizedfthe exish ence of a substance that stimulated 'thefgrowthin' a'syn thetic medium of Leuconostoc citrovorumi "This unknown substance was found to-be'p'resent in commercial liver extracts and also inliver and a .wide variety ofnatural materials. It has subsequently been found that thelunknown substance can replace folic acid vrequirements in; micro-organisms andchicksl- .Ithasl further been found that the growth factor will. reverse the action of pteroylglutamic acid antagonists and, surprisingly, will'reverse. the toxic effects of 4-aminopteroylglutamic acid or aminopterin (N- [4-{ (2,4-diamino-6-pyrimido[4,i-blpyraayl.)- methyl] -amino}benzoyl]glutainic acid) in mice and bacteria under conditions in which pteroylglutamic acid is inelfective. 4 j I In .our copending application, Serial No 7 October 23, 1950, now abandoned, we' described'cer'tain substituted pteridines having in the 4-position an amino radical which had physiological activity. We have now found that other substituted ptetidines hei'tainafter'described having an amino group in theg4-position have ac tivity in inhibiting growth of cert-ainrbacteria andthere fore may find use in medicine. These compounds are 'described hereinafter in the present application, whichis-a continuation-in-part of said application, Serial 1 No. 191,722. I Y

The new compounds of the present invention may be prepared by reduction of the formyl derivatives of certain known compounds which have been previously prepared, some of which have been described in the literature, and subsequent treatment under neutral or alkaline conditions. The starting materials that may be used in the process of the present invention to prepare the new compounds may be represented by the following structural formula:

M E}. g XE\}1I/\I\3I/7 I R: in which X is amino, :1 monoalkylamino or a dialkylamino radical, R is a hydroxyl group or a=radical of an amino acid and R1 and R2 are hydrogen or halogenra'di cals. The compounds described above are formylated by reacting with formic acid, an alkyl, for'rniminowether or other compounds capable of introducing the formyl 'radical into the N -position of the above compounds.

In the above formula R is described as a 'hydroxyl group or a radical of an amino acids. This amino acid is prefer-- ably glutamic acid or a peptide of glutamic acid such as glutamylglutamic acid, glutarnylglutamylglutamic acid. hexa-glutamylglutamic acid, .glycyl glutamic acid or the. like. The amino acid radical may, however,-be-ofanother amino acid, for example, aspartic acid, glycine, alanine,

tinned;

'least one of which 2,742,468 Patented Apr. .17, 1956 dl-isoleucina- .dl-valine, serine, or the like.

salts of these compounds may also be used inthe process if desired.

The compounds of the present invention can also be pr epared'by reduction of the2-substituted-4raminopteridine followed .by formylation. I I

The exact structure of the new compounds produced by the reduction of the above formyl compounds and subsequent treatment under alkaline conditions has not been definitely determined as yet in view of their complex nature. It is believed, however, that they may be repre-, sented by the following formulae: (1), NH: Y In femw- P- l p \N N R2 H in which Yan'dY are hydrogen or a formyl radical, at

is aformyl radical, and after treatment under alkaline conditions by the following formula:

O=CH I In above structural formulae X, R, R -i- 'andRz are as defined above. "It will also be understood that the above may exist in tautomeric forms, depending upon the conditions in which they are present. 2

The reduction of the formyl compound by which the newproduc't of the present invention are preparedmay be; conducted either with or without theaid of catalysts. When using aycatalytic reduction a wide variety of solventsmay be used in the process including formic acid,

Y alcohols, glycol, acetic acid'and others, depending upon the nature of the catalytic agent. The 'presenceof small amounts of water is not objectionable. The temperature of the reduction may occur over a wide range of temperatures from about 0 C. to about 150 C.

Many different reduction catalysts may be employed in the process such as platinum black, palladium on charcoal, Raney nickel, and others hereinafter described.

"In general, the reaction occurs quite rapidly and appre ciable yields of the tetrahy-dro pteridine can be obtained in as little time as ten minutes at room temperatureusing, f fo'rexa'mple, platinum in formic acid. The reduction is usually complete within a periodof 30 minutes to about 2 hours.

'In the process of the present invention the tetrahydro I derivatives of the 2-substituted-4-amino-N -formylpteroic 3 an aqueous medium-is preferable although mixtures ofacid oramino acid amides thereof or their t-automeric ring forms and N N -diforrnyl- 2-substituted-4-aminopteroic acid or amino acid amides thereof, prepared as described above, are treated with alkaline agents in an essentially aqueous medium at a pH of 7 to about pH 14. Alkaline agents such as alkali metal hydroxides, carbonates, bicarbonates and phosphates and the alkaline earth metal hydroxides and quaternary ammonium bases are useful and operable to produce the desired range. The use of watervand other water miscible solvent can be used sucha v alcoholic mixtures. room temperature'on Flong standing. It can be madeto' take place in a shorter period of time such as 30 minutes to 2 or 3 hours by heating at'a temperature of C. to

p v Since the amino acids contain carboxylic acid groups, obviously.

The reaction occursi slowly at:

The generally preferred procedure of preparing the new compounds of the present invention is as follows: The 4-amino substituted pteridines such as described hereinbefore are first formylated in 90-100% formic acid at -100 C. The formyl compounds thus prepared may be isolated if desired and the reduction completed in a different solvent. However, it is generally more convenient to continue the reduction in the same solution by adding the catalyst to the solution after which hydrogen may be passed into the reaction mixture with shaking or stirring. In some cases it may be advantageous to activate the catalyst with hydrogen before mixing with the formylated substituted pteridine, although this procedure is not necessary. The pressure of hydrogen in the reaction vessel may be from 1 to 100 atmospheres or more but under ordinary conditions 2 or 3 atmospheres pressure are sufiicient to obtain a good yield. After the reduction, the catalyst is removed by filtration and the solution is buffered in an aqueous alkaline medium, preferably at a pH of 7 to 14 such as an aqueous solution of sodium bicarbonate. When desirable, to remove the excess formic acid from the reduction solution after removal of the catalyst, the solution can be poured into ether and the insoluble product, the N -formyl derivative, isolated by filtration, or the solvent may be removed by distillation and the residue recovered; and subsequently treated under alkaline conditions to yield the N -formyl derivatives.

The reduced formyl substituted pteridine, prepared as described above, may be absorbed on various absorbing agents such as charcoal, magnesium silicate, etc. and these procedures may be used to purify the crude reaction products by chromatographic absorption methods.

The following examples describe various processes for obtaining the biologically active substances of the present invention by the reduction of certain formyl substituted; pteridines. All parts are by weight unless otherwise indicated.

Example 1 Two parts of 4-aminopteroylglutamic acid of 73% purity are heated for one hour on the steam bath in 40 parts of 98-100% formic acid, and the solution is reduced for two hours in the presence of 0.50 part of platinum oxide catalyst at 35 lbs. hydrogen pressure. The platinum is filtered OE and the solution poured into sodium bicarbonate solution. The product shows growth inhibition activity against certain bacteria.

Example 2 The process of Example 1 is repeated with the excep-- tion that the 4-aminopteroylglutamic acid is stirred for two hours in 98100% formic acid at 2025 C. The resulting solution, upon reduction and isolation of the product as described in Example 1, results in a product with similar growth inhibition activity against certain bacteria.

Example 3 0.5 part of 2-dimethylamino-4-aminopteroylglutamic acid are dissolved in 20 parts of 98-l00% formic acid and heated one hour on the steam bath. After cooling to room temperature, 0.10 part of platinum oxide catalyst is added and the mixture is reduced at 35 lbs. hydrogen pressure for two hours. tered off and the solution poured into 300 parts of water containing an excess of sodium bicarbonate. The solution is clarified and adjusted to pH 4, followed by the addition of parts of activated charcoal. After stirring for one-half hour at room temperature, the mixture is filtered, and the charcoal cake extracted with an ammonia-ethanol mixture. This is then distilled to dryness and the residue triturated with an ethanol-ether mixture.

A dull yellow solid ammonium salt of tetrahydro 2-di-' methylamino-4-a1nino5-formylpteroylglutamic acid separates, which is then filtered off and dried under reduced The platinum is filpressure and weighs 0.15 part. It possesses growth inhibiting activity.

Example 4 One part of 3,5-dichlor&4-aminopteroylglutamic acid is dissolved in 20 parts of 98100% formic acid and heated on the steam bath 45 minutes, giving a clear yellow solution. After cooling to room temperature, 0.20 part of platinum oxide catalyst is added and the mixture is reduced at 35 lbs. hydrogen pressure for one and one-half hours. The platinum is filtered oif and the solution poured into 300 parts of water containing an excess of sodium bicarbonate. It is then adjusted to pH 4, 10 parts of activated charcoal added and the mixture stirred'for one-half'hour. The activated charcoal is then filtered off and extracted with an ammoniaethanol mixture... The resultant solution is distilled under reduced pressure to dryness and the residue taken up in 10 parts of water, yielding a clear solution. This is adjustedto pH 7 and a concentrated solution containing 0.4 part of barium chloride added, followed by 40 parts of ethanol. A white barium salt of tetrahydro 3,5Gdichloro-4-amino-5-formylpteroylglutamic acid is precipitated; dry weight, 0.51 part. It inhibits the growth of certain bacteria.

' Example 5 This reaction is carried out in the same manner as describedin Example 4. From one part of 4-aminopteroyla'minomalonic acid, 0.57 part of the yellow barium saltof tetrahydro-4-amino-5-formylpteroy1aminomalonic acid is obtained. It possesses growth inhibiting action against bacteria.

' a I Example 6 This reaction is carried out in the same manner as described in' Example 4. From one part of 4aminopt er'oyldl-isoleucine there is obtained 0.17 part of the yellow barium salt of tetrahydro-4-amino-5-formylpteroyl-dl isoleucine. It possesses growth inhibitory action. 7

Example 7 This reaction is carried out in the same manner as described in Example 4. From one part of 4-aminopteroyl-dl-valine there is obtained 0.35 part of the yellow barium salt of tetrahydro-4-amino-5-forrnylpteroyl-dlvaline. It possesses growth inhibitory action.

We claim:

1. Compounds of the group consisting of thosehaving the structure:

in which X is a member of the group consisting of amino, mono-alkyl-arnino and dialkylamino radicals, Y and Y are members of the group consisting of hydrogen and formyl, at least one of which is a formyl radical,

o JLR is a member of the group consisting of carboxylic acid, carbonylaspartic acid, carbonylglutamic acid, carbonylglntamylglutamic acid, carbonylglutamylglutamylglutamic acid, carbonylaminomalonic acid, carbonylvaline and carbonylisoleucine and R1 and R2 are members of the group consisting of hydrogen and chlorine radicals, and therapeutically useful cationic'salts thereof.

2. Tetrahydro-2-dialkylamino-4-amiuo-S-formylpteroylglutamic acids.

3. Tetrahydro-4-amino-5-formylpteroylglutamic acid. 4. Tetrahydro-2-dimethylamino 4 amino 5 formylpteroylglutamic acid.

5. Tetrahydro 3',5-dichloro-4-amino-S-formylpteroylglutamic acid.

6. Tetrahydro-4-amino 5 formylpteroylaminomalonic acid.

7. Tetrahydro-4-amino-5-formylpteroyl-dl-isoleucine. 5

8. A process which comprises subjecting compounds of the general formula:

I N TCHr-N- o-n K /L R2 in which X is a member of the group consisting of amino, mono-alkyl-amino and dialkylamino radicals,

O i-R is a member of the group consisting of carboxylic acid,

.carbonylaspartic acid, carbonylglutamic acid, carbonyl- 9. A process in accordance with claim 8 in which said compounds are hydrogenated with platinum and hydrogen until two moles of hydrogen are absorbed.

10. A process which comprises subjecting 4-amino-l0- formylpteroylglutamic acid to the action of hydrogen and a metal catalyst until two moles of hydrogen are absorbed, subsequently treating the reaction product under alkaline conditions and recovering tetrahydro-4-amino-S-formylpteroylglutamic acid therefrom.

11. A process which comprises subjecting a 2-dialkyl amino-4-amino-10-formylpteroylglutamic acid to the action of hydrogen until two moles of hydrogen are absorbed and subsequently treating the reaction product under alkaline conditions and recovering a tetrahydro-Z-dialkylamino-4-amino-5-formylpteroylglutamic acid therefrom.

12. A process which comprises the step of hydrogenating 4-arnino-10-formylpteroylglutamic acid by the action of platinum and hydrogen until two moles of hydrogen are absorbed, and recovering tetrahydro-4-amino-10-formylpteroylglutamic acid therefrom.

13. A process which comprises hydrogenating 2-dimethylamino-4-arnino-10-formy1pteroylglutamic acid by the action of platinum and hydrogen until two moles of hydrogen are absorbed, treating the reaction product under alkaline conditions and recovering tetrahydro-Z-dimethylamino-4-arnino-5-formylpteroylglutamic acid therefrom.

14. A process which comprises hydrogenating 3',5-dicl1loro-4-amino-10-formylpteroylglutamic acid by the action of platinum and hydrogen, until two moles of hy drogen are absorbed, subsequently treating the reaction product under alkaline conditions and recovering tetrahydro 3,5 dichloro -4- amino -5- formylpteroylglutamic acid therefrom.

15. A process which comprises the stepof hydrogenating 4-amino-l0-formylpteroylaminomalonic acid by the action of platinum and hydrogen until two moles of hydrogen are absorbed and recovering tetrahydro-4-amino- 10-formylpteroylaminomalonic acid therefrom.

16. A process which comprises the step of hydrogenating 4-amino-10-formylpteroyl-dl-isoleucine by the action of platinum and hydrogen until two moles of hydrogen are absorbed and recovering tetrahydro-4-amino-10-formylpteroyl-dl-isoleucine therefrom.

No references cited. 

1. COMPOUNDS OF THE GROUP CONSISTING OF THOSE HAVING THE STRUCTURE:
 8. A PROCESS WHICH COMPRISES SUBJECTING COMPOUNDS OF THE GENERAL FORMULA: 